New chemotherapeutics are urgently required to control the tuberculosis pandemic, which is fueled by the emergence of multi-drug- and extensively-drug-resistant Mycobacterium tuberculosis strains and the bacterium's catastrophic alliance with HIV. In addition to genetic resistance to drugs, M. tuberculosis cells can become persistors, cells that are phenotypically resistant to the killing by drugs. This ability to enter into a persistent state necessitates long periods of chemotherapy and represents the greatest challenge in improve TB chemotherapies. Unfortunately, little is known about the metabolic state of persistors. To elucidate the physiological state of persistent M. tuberculosis cells, we have used mass spectroscopy to generate metabolic profiles of M. tuberculosis when grown in aerobic and hypoxic growth conditions (a persistence model) and found several key control points modulating NADH/NAD (redox) metabolism. To further characterize the metabolic profiles, we have investigated the Tricarboxylic Acid Cycle (TCA) of M. tuberculosis. Using a combination of genetic and biochemical approaches, we have discovered that M. tuberculosis possesses a set of genes encoding ketoglutarate oxidoreductase, a key enzyme of the TCA that is generally observed in anaerobic bacteria. In addition, we have discovered that mutations in the gene encoding NADH dehydrogenase II (ndh) confer resistance to Isoniazid and Ethionamide. By combining metabolite profiling, and stable isotope flux profiling, with mutational studies we intend to characterize persistent states of M. tuberculosis. This knowledge should lead to novel strategies to effectively kill persistent M. tuberculosis cells. PUBLIC HEALTH RELEVANCE: New chemotherapeutics are urgently required to control the tuberculosis pandemic, which is fueled by the emergence of multi-drug- and extensively-drug-resistant Mycobacterium tuberculosis strains and the bacterium's catastrophic alliance with HIV. In addition to genetic resistance to drugs, M. tuberculosis cells can become persistors, cells that are phenotypically resistant to the killing by drugs. This proposal seeks to develop new understandings of persisting M. tuberculosis cells providing the knowledge for novel and shortened chemotherapies.